Acinetobacter baumannii is an important nosocomial pathogen that causes a range of infections, including respiratory and urinary tract infections, meningitis, endocarditis, wound infections, and bacteremia. In fact, A. baumannii is now responsible for up to 20% of all intensive care unit infections in some regions of the world with pneumonia being the most common presentation. Additionally, A. baumannii is a considerable cause of infection in combat soldiers and is the most common gram-negative bacillus recovered from traumatic injuries to extremities obtained on the battlefield. The clinical significance of A. baumannii has been propelled by this organism's rapid acquisition of resistance to virtually all antibiotics. Taken together, these facts have established A. baumannii as a significant threat to the health of Veterans. Based on this, we have recently initiated a research program focused on identifying novel targets for therapeutic intervention against A. baumannii. In this application, we describe our discovery of an immune enhancing biologic that has tremendous therapeutic efficacy against A. baumannii pneumonia and can cure this infection in mice. We have made the exciting discovery that transposon mutagenesis of A. baumannii severely attenuates its virulence, regardless of the site of transposon insertion. Moreover, strains that have experienced transposon mutagenesis are capable of curing infections caused by wildtype A. baumannii during co-infection experiments, and this attenuating phenotype does not require that the transposon mutagenized strain is viable. This attenuation requires exposure of a DNA-protein complex on the surface of A. baumannii, and relies on key innate immune signaling pathways in the host. Finally, we have found that transposon mutagenesis of A. baumannii leads to up-regulation of genes encoding for the Type IV secretion system (T4SS) and increased abundance of pilus-like surface appendages visible by electron microscopy. Based on these findings, we propose a model whereby A. baumannii up-regulates its T4SS upon exposure to exogenous DNA, and this secretion system is recognized by the innate immune system of the host to coordinate clearance of the invading pathogen. To test this model we propose a series of three integrated Specific Aims. In Aim 1 we will define the immune response elicited by A. baumannii T4SS hyperexpressors. Results obtained from these experiments may lead to the rational design of immunomodulatory therapies for the treatment of bacterial pneumonia. In Aim 2 we will elucidate the mechanism by which transposition alters T4SS expression. In these experiments we will determine the mechanism by which the T4SS is up-regulated upon exposure to foreign DNA and interrogate the impact of the A. baumannii T4SS on DNA transfer and exchange. These results will lay the foundation for studies focused on reducing DNA transfer and preventing the acquisition of antimicrobial resistant determinants by this organism. Finally, in Aim 3 we will determine the broad spectrum efficacy of T4SS-based immune enhancing biologics. A. baumannii transposon mutants exhibit therapeutic efficacy against pneumonia caused by Pseudomonas aeruginosa suggesting that this strategy has broad applicability against a variety of infectious agents. In addition to the potential clinical benefits of this discovery, these proposed experiments will provide us with a tool to study the host-pathogen interaction during infection with the goal of defining a successful immune response to pneumonia caused by Gram negative pathogens.